There is an increasing need for rapid, small scale and highly sensitive detection of biological molecules in medical, bioterrorism, food safety, and research applications. Nanostructures such as silicon nanowires and carbon nanotubes display physical and electronic properties amenable to use in miniature devices. Carbon nanotubes (CNTs) are rolled up graphene sheets having a diameter on the nanometer scale and typical lengths of up to several micrometers. CNTs can behave as semiconductors or metals depending on their chirality. Additionally, dissimilar carbon nanotubes may contact each other allowing the formation of a conductive path with interesting electrical, magnetic, nonlinear optical, thermal and mechanical properties.
It is known that single-walled carbon nanotubes are sensitive to their chemical environment, specifically that exposure to air or oxygen alters their electrical properties (Collins et al. (2000) Science 287:1801). Additionally, exposure of CNTs to gas molecules such as NO2 or NH3 alters their electrical conductance (Kong et al. (2000) Science 287:622). Thus chemical gas sensors can be designed on the basis of the electrical properties of carbon nanotubes such as described in DE10118200.
Sanjay and Kramer ((1996) Nature Biotech. 14:303) describe the detection of DNA in solution using molecular beacons. These are stem-loop structures that contain a fluorescence emitter and quencher, one on each strand at the base of the stem, that open in the presence of a DNA single strand or RNA, complementary to the loop region, producing an increase in the fluorescence yield of the emission. Used for real-time PCR, these structures produce a dequenching of one fluorescence emitter for every complementary nucleic acid strand hybridized.
In WO 02/48701 articles are described that use nanowires, including CNTs, to detect different types of analytes including biological analytes. The nanowire may be modified by attaching an agent that is designed to bind an analyte, the binding to the nanowire or to a coating on the nanowire then causes a detectable change in conductance. In this detection system, the interaction between the binding agent and the analyte to be detected alters the electrical conductance of the nanowire. This requirement in turn limits the functional location of the binding agent with respect to the nanowire in that they must be in close proximity, 5 nanometers or less.
Carbon nanotubes have been used in electrocatalysis. Microelectrodes constructed of multiwalled carbon nanotubes were shown to provide a catalytic surface for electrochemical reduction of dissolved oxygen, potentially useful in fuel cell applications (Britto et al. (1999) Advanced Materials 11:154). A film of single walled carbon nanotubes functionalized with carboxylic acid groups on a glassy carbon electrode showed electrocatalytic behavior with several redox-active biomolecules, involving reduction of the carboxylic acid groups (Luo et al. (2001) Anal. Chem. 73:915). Toluene-filled multiwalled carbon nanotubes as a film on a gold electrode surface were shown to respond better to electroactive biomolecules than empty carbon nanotubes (Zhang et al. (2003) Electrochimica Acta 49:715).
In WO 2004/034025 a system to measure the redox potential is described that uses a potentiometric electrochemical system based on a metal-coated silicon nanowire.
There is a need for a nanoscale detection system that has the ability to indirectly detect an analyte in a solution-based format that can provide a signal whose concentration greatly exceeds the concentration of the analyte. Applicants have solved this problem by developing a carbon nanotube based nanosensor that responds to a target analyte by altering the redox potential of a redox effector in solution, which in turn alters the redox state of the CNT and causes a change in its conductance. The concentration of redox-active effector molecules may far exceed that of the analyte. The assay is accomplished in solution and eliminates the need for immobilization of the analytes for detection.